Perfusion-decellularized pancreas as a natural 3D scaffold for pancreatic tissue

and whole organ engineering

Saik-Kia Goh1, Suzanne Bertera2, Phillip Olsen1, Joe Candiello1, Willi Halfter3, Guy Uechi4, Manimalha Balasubramani4, Scott Johnson8, Brian Sicari6, 8, Elizabeth Kollar8, Stephen F. Badylak1, 5, 8, Ipsita Banerjee1, 6, 7

1Department of Bioengineering, University of Pittsburgh2Division of Immunogenetics, Department of Pediatrics, Children’s Hospital of Pittsburgh3Department of Neurobiology, University of Pittsburgh4Genomic and Proteomic Core Facility, University of Pittsburgh5Department of Surgery, University of Pittsburgh6Department of Pathology, University of Pittsburgh7Department of Chemical and Petroleum Engineering, University of Pittsburgh8McGowan Institute for Regenerative Medicine, University of Pittsburgh

ABSTRACT:

Approximately 285 million people worldwide suffer from diabetes, with insulin

supplementation as the most common treatment measure. Regenerative medicine

approaches such as a bioengineered pancreas has been proposed as potential

therapeutic alternatives. A bioengineered pancreas will benefit from the development of

a bioscaffold that supports and enhances cellular function and tissue development.

Perfusion-decellularized organs are a likely candidate for use in such scaffolds since

they mimic compositional, architectural and biomechanical nature of a native organ. In

this study, we investigate perfusion-decellularization of whole pancreas and the

feasibility to recellularize the whole pancreas scaffold with pancreatic cell types. Our

result demonstrates that perfusion-decellularization of whole pancreas effectively

removes cellular and nuclear material while retaining intricate three-dimensional

microarchitecture with perfusable vasculature and ductal network and crucial

extracellular matrix (ECM) components. To mimic pancreatic cell composition, we

recellularized the whole pancreas scaffold with acinar and beta cell lines and cultured

up to 5 days. Our result shows successful cellular engraftment within the decellularized

pancreas, and the resulting graft gave rise to strong up-regulation of insulin gene

expression. These findings support biological utility of whole pancreas ECM as a

biomaterials scaffold for supporting and enhancing pancreatic cell functionality and

represent a step toward bioengineered pancreas using regenerative medicine

approaches.

Keywords:

Whole organ decellularization; Extracellular matrix scaffold; Tissue and organ

engineering; Pancreatic β-cells

1. INTRODUCTION

Diabetes mellitus is a global disease with immense economic and social burden.

According to the World Health Organization, at least 285 million people worldwide suffer

from diabetes [1]. While pharmaceutical interventions and insulin supplementation are

the most commonplace treatment of diabetes, these do not represent a cure and can

potentially lead to long term complications [2]. Advances in tissue engineering have

facilitated the development of replacement tissues or organs for the treatment of injured

or degenerative soft tissue [3-5]. The development of a bioengineered pancreas by

appropriate combination of cells, biomaterial scaffolds and biologically active molecules

could provide an alternative avenue for diabetes therapy.

Three-dimensional (3D) scaffold plays a critical role in regenerative medicine and tissue

engineering. For example, cell-scaffold interactions are important for the regulation of

cellular behavior, including pancreatic islet-cell survival and insulin production through

integrin-mediated activation and downstream signaling events [6, 7]. Native tissues and

organs are comprised of unique extracellular matrix (ECM) compositions,

microstructures and biomechanical properties, which maintain distinct signals for

resident cells [8, 9]. Ideally, a tissue engineered scaffold would provide the same or

similar microenvironmental niche to the seeded cells as that of a native ECM.

Therefore, at present, the development of pancreatic tissue engineering scaffolds have

focused on recreating milieu similar to the native islet and surrounding ECM

(summarized in review by Cheng et al. [10] and Candiello et al. [11]). Various synthetic

and natural biomaterials have been exploited in this field with the objective to restore

the critical signal loss between native islet cells and their ECM environment following

islet isolation. Most of these tissue engineering scaffolds, however, use a purified single

ECM protein and do not mimic the intricacy of the pancreatic ECM composition and

ultrastructure. Recently there has been a shift towards multiple-matrix systems of

purified proteins, which have been shown to improve stem cell proliferation and

differentiation [12, 13] and promote isolated islet and β-cell survival and function [14,

15]. Natural ECM derived from native tissues and organs can meet this requirement by

providing a more physiologically relevant scaffold that recapitulates the complex in vivo

microenvironment.

The novel concept of whole organ perfusion decellularization has been described

recently to generate native ECM scaffolds from complex organs such as heart [16-19],

liver [20-23], lung [24-29], kidney [30-32] and more recently pancreas [33-36]. Whole

organ acellular matrices provide an attractive scaffold for the repopulation with cells for

an engineered tissue/organ because of the physiological resemblance of the original

tissue, including intact 3D anatomical architecture, preserved spatial array of ECM

components, vascular network, and biomechanical properties. Prior studies have shown

the positive effect of endothelial cell derived ECM for the enhancement of islet and β-

cell attachment and proliferation [37, 38]. Other works have also shown improved islet

functionality when cultured on decellularized matrices derived from small intestinal

submucosa (SIS) [39, 40] and pancreatic slices [36, 41]. Decellularized pancreas work

described by De Carlo et al. [41] and Mirmalek-Sani et al. [36] revealed the benefits of

2D decellularized pancreas slices to support and maintain islet functions. These initial

studies clearly motivate the need and indicate the potential benefits that can be attained

by a whole organ 3D reconstruction of pancreas. The importance of 3D scaffold is well

recognized for a better representation of the complex in vivo microenvironment and is

important for proper regulation of cell behavior [42], yet recellularization of whole

pancreas 3D scaffold remains to be demonstrated. We hypothesize that the optimal

scaffold to support pancreatic tissue engineering may be the natural 3D ECM scaffold of

the whole pancreas. To this effect, we propose that perfusion-decellularization of whole

pancreas yields an acellular scaffold suitable for pancreatic tissue and organ

engineering.

The present study investigated the possibility of generating an acellular whole pancreas

scaffold by a perfusion decellularization technique and utilizing this biomaterial as a

scaffold to support pancreatic tissue engineering and whole organ regeneration. Toward

this end, we characterized the resultant native pancreatic ECM-scaffold for preservation

of ECM composition, 3D structural integrity, and biomechanical properties. We then

repopulated the decellularized pancreas with endocrine and exocrine cell types to

demonstrate the feasibility of recellularization. Furthermore, the effects of pancreas

derived ECM scaffold on modulating cellular functionality such as insulin gene

expression was determined.

2. METHODS

2.1 Mouse pancreas harvest and cannulation

All animal work performed was in accordance with animal welfare act, institutional

guidelines and approved by Institutional Animal Care and Use Committee of the

University of Pittsburgh). Female ICR mice (Taconic), between the ages of 6-12 weeks

were euthanized by CO2 inhalation followed by cervical dislocation. Sterile conditions

were observed when removing the pancreas. A laparotomy was performed and a 24 G

catheter was inserted approximately 1 cm into the anterior hepatic portal vein and

sutured in place. The distal end of the superior mesenteric vein and large branches of

splenic arteries and veins were carefully ligated to prevent leakage. The pancreas was

then carefully dissected free from all adjacent structures including the stomach,

intestine, spleen and mesenteric tissue.

2.2 Perfusion decellularization of mouse pancreas

The isolated pancreas was connected to a perfusion system to allow retrograde

perfusion at 8ml/min, in which solutions flowed through the hepatic portal vein, into the

splenic vein, and throughout the vasculature of the pancreas. Next, ionic detergent,

0.5% SDS (Invitrogen) in deionized water was used as perfusate to rinse cells and cell

debris out of the pancreas. After the tissues became translucent (about 325 min),

subsequent steps of perfusion were 15 min of deionized water perfusion and 15 min of

1% Triton X-100 (Sigma Aldrich) in deionized water. A solution of benzonase (90U/ml,

Sigma) was perfused for 15 min and a final washing step of 10% fetal bovine serum

(FBS, Life Technologies) in PBS with Pen/Strep (100U/ml) perfused the pancreas for

additional 48 hours to clear remaining cellular debris.

2.3 Cell culture

The AR42J acinar cell line (CRL-1492, ATCC, VA, USA) was used at passage 18-25.

AR42J was cultured inF-12K medium (ATCC, VA, USA) supplemented with 20% FBS

(Life Technologies) and 100 U/ml penicillin/streptomycin (Life Technologies) on T75

tissue culture flasks. The MIN-6 β-cell was used at passage 19-26. MIN-6 was cultured

in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 15%

FBS (Life Technologies) and 100 U/ml penicillin/streptomycin (Life Technologies) on

T75 tissue culture flasks. Both cell types were cultured at 37°C and in a 95% air / 5%

CO2 atmosphere.

2.4 In vivo implantation of decellularized pancreas

All animal work was approved by the Institutional Animal Care and Use Committee of

the University of Pittsburgh. For preliminary biocompatibility studies, a 1 cm2 perfusion-

decellularized pancreas construct was surgically placed within a dorsal subcutaneous

pocket of an adult mouse. Female C57BL/6 mice, age 6–8 weeks, were purchased from

Jackson Laboratories (Bar Harbor, ME). The mouse was anesthetized and maintained

at a surgical plane of anesthesia with 1.5–2.5% isoflurane in oxygen and positioned in

dorsal recumbency. The surgical site was prepared in sterile fashion using 70%

isopropyl alcohol followed by the placement of sterile drapes. For the subcutaneous

surgical implantation: A central longitudinal incision measuring approximately 1.5 cm in

length was made in the epidermis, dermis, and fascia to expose the underlying muscle

tissue on the dorsal side. Dermal layers and underlying connective tissue was

undermined to create a pocket with similar size to the implant construct. The

decellularized pancreas construct was sutured to the underlying muscle with four non-

absorbable marking sutures (3-0 prolene) and the skin was closed over the implant site

in uninterrupted fashion. A minimal amount of suture material was placed only at the

construct corners to avoid eliciting a host response to the suture that would obscure the

host response to the implanted material. The incision was covered in betadine ointment

after closure and assessed for signs of infection for2 days postsurgery. The mouse

received Buprenex (buprenorphine hydrochloride, 0.25 mg/kg) for analgesia, and Baytril

(enrofloxacin, 20 mg) an antibiotic, for 3 days postoperatively. The animal survived the

surgical procedure and its predetermined study period without complications. After 14

days the mouse was euthanized with 5% isoflurane in oxygen followed by an

intracardiac injection of potassium chloride to induce cardiac arrest. Following

euthanasia the implant along with the surrounding skin and muscle were harvested and

fixed in 10% NBF for histologic evaluation.

2.5 Recellularization and culture of seeded-pancreatic construct

MIN-6 cells (30x106) were trypsinized and diluted in 3mL of medium. The cell

suspension was introduced into the decellularized pancreas by means of retrograde

gravity perfusion via the hepatic portal vein in 3 steps, 1mL each, with 20 min interval

between each step. MIN-6 seeded pancreas was immersed and cultured in the same

MIN-6 cell culture medium described above for 5 days. For co-recellularization strategy,

AR42J was used as the second cell type for the recellularization. Similarly, AR42J

(30x106 cells) were trypsinized and diluted in 3mL of medium. The AR42J cell

suspension was inoculated into the pancreas by retrograde perfusion through the

pancreatic duct but the MIN-6 cells were introduced through the vasculature. The two

cell types were seeded in sequence, with the same multi-step repopulation mentioned

for single-cell type seeding. Cells were allowed to attach for 2 hours before introducing

a gentle rinse through both the hepatic portal vein and pancreatic duct in order to wash

out any unattached cells. The cell-seeded pancreatic construct was left undisturbed and

submerged in co-recell culture medium (DMEM/F12 supplemented with 15% FBS and

100 U/ml Pen/Strep). Both cannulation to the portal vein and pancreatic duct were kept

intact to allow perfusion feeding of media every day. The seeded pancreas was cultured

under static conditions at 37°C with a 95% air / 5% CO2 atmosphere for 5 days.

2.6 MIN-6 seeded on different ECM substrates and 2D thin slices of decellularized

pancreas

Growth factor-reduced Matrigel (BD) was used as a thin coating according to

manufacturer’s instruction. Collagen IV (BD) and fibronectin (Milipore) were used at a

concentration of 5 mg/ml. These ECM substrates were used for coating non-tissue

culture-treated 12-well plates (BD). Collagen type I (BD) was used as 5 mm thick gels.

Coated and untreated (UT) plates were blocked with 1% BSA (Sigma). Control culture

was conducted on UT plates blocked with 1% BSA (Sigma). To prepare thin

decellularized pancreata slices, decellularized pancreata were filled with 1.1% alginate

solution, and submerged in 100mM Calcium Chloride solution to crosslink the alginate.

The solidified pancreas was sliced with a tissue slicer (Thomas Scientific) to yield thin,

uniform, transverse sections. The alginate within the thin slices of pancreas were

dissolved in a sterile dissolution buffer consisting of 50 mM tri-sodium citrate dihydrate

(Sigma), 77 mM sodium chloride (VWR International, Leicestershire, UK), and 10 mM

HEPES (Sigma) in PBS for 15–20 min while stirring gently at room temperature.

Finally, the thin slices of pancreas were rinsed and covered with sterile PBS with

Pen/Strep (100U/ml), and placed at 4°C until use. Cloning cylinder (Fisher) was applied

and sealed with sterile silicone grease (Dow Corning) to the pancreatic slice to

consolidate the seeded cells only to the pancreatic slice. MIN-6 cells (25,000 cells/cm2)

were cultured on these various different ECM substrates for 5 days before harvest for

qPCR analysis.

2.7 Two-photon microscopy

Two-photon microscopy was performed with an upright Olympus FV1000 MPE multi-

photon microscope (Olympus, Central Valley, PA, USA) and a Mai Tai DeepSee

femtosecond-pulsed laser (Spectra-Physics, Santa Clara, CA, USA) tuned at 800 nm.

The pancreatic slice was line-scanned and fluorescence emission was captured by

three non-descanned external photomultiplier tube (PMT) detectors coupled to the

following long-pass dichroic mirrors and band pass emission filters: 505 nm mirror and

460–500 nm filter (blue channel), 570 nm mirror and 520–560 nm filter (green channel)

and 575–630 nm filter (red channel). The pancreatic slice was placed on an imaging

dish having a #1.5 coverslip and immersed in PBS. Fixed xy planes spanning 505 × 375

μm at a resolution of 0.994 μm/pixel and depth of 1–50 μm from the surface of scaffold

were imaged using a high numerical aperture (NA = 1.05), water-immersion 25X

objective.

2.8 Immunohistochemistry (IHC)

Native, decellularized and recellularized pancreas were fixed, sectioned and stained

following protocol previously described [43]. Briefly, tissue samples were fixed with 4%

formaldehyde (ThermoFisher), cryoprotected with 30% sucrose and cut into 7 µm thick

sections. For immunostaining, the following primary antibodies were used: rabbit anti-

laminin, rabbit anti-collagen I, rabbit anti-collagen IV, rabbit anti-fibronectin (Abcam,

1:200), rabbit anti-Cpeptide (Cell signaling, 1:100) and rabbit anti-α-Amylase (Sigma

Aldrich, 1:200). Secondary antibodies used were: donkey anti-rabbit Alexafluor 488

(1:500, Invitrogen) and donkey anti-rabbit Alexafluor 555 (1:500, Invitrogen). For co-

labeling using antibodies from the same host species, sequential staining was

conducted. After the first primary antibody staining, an additional blocking step was

included prior to the addition of a second primary antibody. For actin staining, phalloidin

rhodamine (Cytoskeleton, 1:200) was implemented and stained for 30 min in the dark.

The slides were washed again three times with 1x PBS (5-10 min) each before being

mounted with ProLong® Gold Anti-fade Reagent with DAPI (Invitrogen). Images were

recorded with Metamorph 7.5.6.0 (Molecular Device) on an Olympus IX81 inverted

microscope (Olympus, Central Valley, PA, USA).

2.9 ECM digestion and proteomics analysis

The ECM pellet was digested for 2h with each of the following enzymes: 0.15 mU/µl

heparan sulfate lyase and chondroitin ABC lyase from Flavobacterium heparinum and

Proteus vulgaris, respectively (Seikagaku Corporation, Japan) and washed in PBS. The

ECM proteins were dissolved in 8M urea and 1x reducing SDS sample buffer. The

proteins from the ECM preparation were separated on 3.5-15% gradient SDS PAGE

under reducing conditions [44]. The gel lane was divided into 11 slices and taken

through a typical in-gel digestion procedure. Briefly, gel bands were destained, reduced

with 2.5mM tris (2-carboxyethyl) phosphine and alkylated with 3.75mM iodoacetamide,

followed by in-gel digestion with 13ng/µl of trypsin (Promega) overnight at 370C. Peptide

digests were analyzed by nano Liquid Chromatography-tandem mass spectrometry

(LC-MS/MS), on a Thermo Fisher LTQ OrbitrapVelos connected to a Waters Acquity

UPLC system (Waters Corp., Milford, MA), using a 90 minute gradient. Protein

identification was performed with Proteome Discoverer 1.3 using the Sequest search

engine. Database searches used the Uniprot complete mouse database (downloaded in

December 2012 merged with a contaminant database from ABSciex, 50838 sequences,

24435643 residues). Settings were for a full trypsin digest, with two missed cleavages,

one static modification (cysteine carbamidomethylation), two dynamic modifications

(oxidized methionines and hydroxyproline), mass tolerance of 10 ppm for precursor

mass and 0.5 Da for fragment masses. Percolator, a post-processing software using a

target/decoy database approach, was used to evaluate the accuracy of peptide

identifications. Peptide identifications were filtered with a q-value cutoff of 0.01 (1%

global False Discovery Rate, FDR). Proteins were grouped using the maximum

parsimony principle and this list was imported to ProteinCenter (Thermo) and compared

with the IPI mouse database for statistical analysis to identify over-represented Gene

Ontology terms and functional classification.

2.10 DNA quantification

The decellularized pancreata (n=3) were digested with papain solution at 60°C for 6 h.

The native pancreata (n=3) were digested in papain solution as controls. Papain (Sigma

Aldrich) was dissolved at 400 mg/ml in 0.1 M phosphate buffer (pH 6.0), with 5 mM

cysteine hydrochloride (Sigma Aldrich), and 5 mM EDTA (Sigma Aldrich). The lysates

were used for detection of the DNA and sulfated glycosaminoglycan (sGAG) content. A

DNA quantification kit - Quant-iT™ PicoGreen® dsDNA Assay kit (Invitrogen) was used

to measure DNA content according to manufacturer’s instruction. The fluorescence

reading (excitation: 485 nm and emission: 528 nm) was taken on a plate reader

(Synergy 2, Biotek), and the absolute amount of DNA (ng/mL) was quantified against a

lambda DNA standard curve (0 ng/mL- 1000 ng/mL).

2.11 sGAG content characterization

For qualitative determination of presence of GAG, Alcian Blue staining was done

according to manufacturer’s instruction (Newcomer Supply). Briefly, slides were

deparaffinized and hydrated through graded ethyl alcohols to distilled water. Next, slides

were placed in 3% acetic acid for 3 min and then directly into 1% Alcian Blue solution

pH 2.5 and stained for 30 min in room temperature. The slides were washed in running

tap water for 10 min followed by a distilled water rinse. The counterstaining was done in

Nuclear Fast Red stain for 5 min before rinsing, dehydrated through graded ethyl

alcohol and cleared in xylene. The slides were mounted with xylene mounting medium.

For qualitative measure of sGAG, the method to produce lysate for DNA quantification

was used here as well. A sGAG quantification kit –Blyscan Sulfated Glycoaminoglycan

Assay kit (Biocolor) was used to measure sGAG according to manufacturer’s

instruction. Briefly, the specimen lysate was mixed with Blyscan dye to bind the GAG.

The GAG-dye complex was then collected by centrifugation. After the supernatant was

removed and the tube drained, the dissociation reagent was added. 100 µl solutions

were transferred into a 96-well plate. Absorbance against the background control was

obtained at a wavelength of 656 nm on a microplate spectrophotometer and the GAG

amount was calculated based on a standard curve obtained with the standard GAG

supplied with the kit.

2.12 Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

Native and decellularized pancreata were fixed in 2.5% glutaraldehyde in 0.1 M PBS

(pH 7.4) for 60 minutes. The samples were washed thoroughly in 3 changes 0.1 M PBS

for 15 minutes each. Next, the samples were fixed in 1% OsO4 in 0.1 M PBS for 60

minutes. This was followed by another 3 changes of PBS washing steps for 15 minutes

each. The samples were then dehydrated in gradient series of alcohol for 15 min each.

Additionally, samples were critical point dried and coated with Au/Pd using a

Cressington Coater 108A sputter coater. Electron microscope images were taken using

a Jeol JSM-6335F field emission SEM. For TEM, the tissue sample was fixed in 2.5%

glutaraldehyde in PBS. The tissue sample were post-fixed in 1% osmium tetroxide in

PBS, dehydrated through a graded series of alcohols and embedded in Epon (Energy

Beam Sciences, Agawam, MA). Thin (60-nm) sections were cut using a Reichert

Ultracut S (Leica, Deerborn, MI), mounted on 200 mesh copper grids and

counterstained with 2% aqueous uranyl acetate for 7 min and 1% aqueous lead citrate

for 2 min. Observation was with a JEOL 1011 transmission electron microscope

(Peabody, MA)

2.13 Preparation of pancreatic tissue for atomic force microscopy (AFM) measurement

Both native and decellularized unfixed samples from murine pancreas were

cryoprotected by soaking in 30% sucrose solution in PBS for 24 hours. The entire

murine pancreas was then set in a mold and covered in OCT cold embedding media

and frozen by exposure to dry ice overnight. The OCT block containing the pancreas

was cryosectioned to a thickness of 20 microns and mounted on SuperFrostPlus glass

slides. All samples were thawed in PBS for 30 minutes prior to testing.

2.14 AFM measurement of pancreatic tissue stiffness

AFM force indentation measurements were performed using the MFP-3D Atomic Force

Microscope (Asylum Research, CA, USA), mounted on top of an Olympus IX-71

fluorescence microscope (Olympus, Tokyo, Japan). All force measurements and

analysis was done using the MFP3D software (Asylum Research) built on IgorPro 6

(Wavemetrics) as previously described [45]. For all measurements a glass silica sphere

(radius 3.5 micron) was attached to the tip of a 100 micron silicon nitride cantilever

(Veeco Systems) with a spring constant of ~0.6 N/m. The stiffness of each alginate gel

was measured at n= 16 random locations on each sample. The Sneddon model was

used to determine the gel stiffness from the force indentation plots.

2.15 Quantitative RT-PCR

RNA was extracted using NucleoSpin kit according to the manufacturer’s protocol. The

sample absorbance at 280 nm and 260 nm was measured using a BioRad Smart Spec

spectrophotometer to obtain RNA concentration and quality. Reverse transcription was

performed using ImProm II Promega reverse transcription kit following the

manufacturer’s recommendation. qRT-PCR analysis was performed for insulin markers,

ins1 and ins2.

The cycle number at the threshold level of log-based fluorescence is defined as Ct

number, which is the observed value in most real-time PCR experiments, and therefore

the primary statistical metric of interest. ΔCt is equal to the difference in threshold cycle

for target and reference or control (ΔCt = Cttarget−Ctreference). ΔΔCt is equal to the

difference between ΔCtsample and ΔCtcontrol (ΔΔCt = ΔCtsample−ΔCtcontrol). The fold change of

a target gene is defined by, fold change = 2-∆∆Ct. qRT-PCR analysis was repeated in

triplicate.

2.16 Statistical analysis

Quantification data were expressed as mean ± SD. Significant differences among

groups were determined by Wilcoxon rank-sum test for two-group comparisons or

ANOVA followed by post-hoc analysis for multiple group comparisons. Probability

values at P < 0.05 (*) indicated statistical significance.

3. RESULTS

3.1 Perfusion-decellularization of whole organ pancreas

Isolated mouse pancreata were cannulated and retrograde perfused via hepatic portal

vein with anionic based detergent, 0.5% SDS to remove cellular content. Alternately,

retrograde perfusion via pancreatic duct was also attempted for pancreas

decellularization, however the process took longer and was also less effective in cell

removal (data not shown). Macroscopically, a gradual change of color was observed

during the perfusion decellularization process (Fig. 1A-D). Perfusion was continued until

whole pancreas turned completely translucent (about 325 min) as depicted in Fig. 1D.

This generated an acellular pancreas scaffold while retaining the gross anatomical

structure of the pancreas. Histological examination by H&E staining showed no remnant

cells after the completion of decellularization (Fig. 1E). Immunostaining of two major

constituents in native pancreas – acinar cells (Amylase) and β-islet cells (C-peptide)

confirmed no residual presence of major pancreatic markers in the decellularized

pancreas (Fig. 1F). To further assess the efficacy of decellularization, DNA

quantification was performed using Picogreen assay. Picogreen analysis showed that

DNA content decreased from 7442.3 ± 2450.2 ng/mg dry weight in normal pancreas to

41.3 ± 11.9 ng/mg dry weight in decellularized pancreas (P < 0.01) (Fig. 1G). These

results suggest that perfusion mediated decellularization of pancreas efficiently removes

pancreatic cellular components. Feasibility of this perfusion decellularization technique

in human-sized organ was also demonstrated by decellularization of bovine pancreas

(supplemental 1).

3.2 ECM characterization: immunohistochemistry and mass spectrometry-based

proteomics analysis

To first characterize the decellularized pancreas, immunohistochemical (IHC) staining

was performed for the evaluation of major ECM components’ spatial presence relative

to that of native pancreas. In native pancreas, the ECM is composed primarily of

networks of collagens and other structural proteins filled with a hydrogel of

proteoglycans [46, 47]. IHC staining showed that collagen I, IV, fibronectin and laminin

were detected in the native pancreas (Fig. 2A). After decellularization, all four major

ECM components were preserved, without any detectable DAPI or actin staining (Fig.

2B). This finding suggests complete removal of cellular and cytoskeletal elements while

maintaining the ECM composition. In addition, the resulting ECM structure and fibril

orientation looked identical to that of the native organ (Fig. 2B). The preservation of

sulfated glycosaminoglycan (sGAG) proteins was evaluated qualitatively by alcian blue

staining (Fig. 2C), which demonstrated that sGAG proteins were retained after

decellularization process. In addition, sGAG content was also assessed quantitatively

using a blyscan assay which demonstrated considerable retention of sGAG content in

the decellularized pancreas, amounting to 52% of that in native pancreas (P<0.01) (Fig.

2D).

Insolubility of the ECM is a challenge to characterizing the pancreatic ECM proteome by

mass spectrometry-based proteomics. In order to improve solubility, pancreatic ECM

was deglycosylated to remove GAG side chains of proteoglycans followed by

solubilization in high molar urea. A GeLC-MS/MS proteomics approach was used

whereby the high molecular weight ECM proteins were separated by low percentage 1D

gels, in-gel trypsin digested followed by LC-MS/MS analysis. Database searching used

stringent 1% false discovery rate to report protein identifications. After protein grouping,

a total of 114 unique proteins were identified in the pancreatic ECM (Supplemental table

1), the majority of which belonged to the ECM family based on Gene Ontology (GO)

Cellular localization. The entire list of identified proteins was compared with the

reference IPI mouse database to identify overrepresented GO cellular localization

terms. The topmost GO terms included ECM and basement membrane (data not

shown); suggesting that the decellularization process successfully removes cellular

proteins yet preserves the ECM components. Table 1 is a categorized list of collagens,

laminins, proteoglycans and ECM associated proteins.

3.3 Biophysical characterization: structural and mechanical properties of decellularized

pancreas

Integrity of the vasculature within the decellularized pancreas was analyzed by trypan

blue dye retrograde injection through hepatic portal vein to visualize the continuity of the

circulatory branching (Fig. 3A). Rapid diffusion through the vascular tree was also

observed (Fig. 3A). Transmission electron microscopy (TEM) and scanning electron

microscopy (SEM) were performed to evaluate the effect of the decellularization

procedure on the 3D architecture and ECM micro-structures of the decellularized

pancreas. TEM analysis revealed intact basement membrane and organized fibers of

collagen preserved upon decellularization (Fig. 3B-C). SEM analysis of the cross-

sectional images of the decellularized pancreatic constructs revealed that large ductal

structures were retained whereas the microvilli cells lining the ducts were removed

(Fig.3D-E). Nanofibrous structures of the ECM were well retained within the

parenchyma (Fig. 3F-G). Hollow and void spaces observed within the decellularized

pancreas are likely attributes of the “footprints” left from pancreatic cells removed during

the decellularization process (Fig. 3H). The recognizable scalloping pattern of arteries

also indicates the preservation of vascular structure likely branched from the greater

pancreatic artery (Fig. 3I). These results suggest that perfusion decellularization of the

pancreas adequately retained the ultrastructure and architecture of the native pancreas.

Biomechanical characteristics of the decellularized pancreas were analyzed using AFM.

Representative cantilever displacement/sample deflection curves (Fig. 4A) shows that

there was a discernible difference between the native and decellularized pancreas, with

the stiffer decellularized sample having a much steeper slope. Young’s modulus as

determined from the AFM curves was found to be 1210±77 Pa for native mouse

pancreas, while the Young’s modulus of decellularized pancreas is on average 3 times

greater (3736±1893 Pa) (Fig. 4B).

3.4 In vivo response to decellularized pancreas

Pancreas ECM was implanted subcutaneously in order to preliminarily evaluate in vivo

biocompatibility in a mouse model. At 14 days post-surgery, histological analysis of the

subcutaneous implantation site showed the presence of mononuclear cells surrounding

the partially degraded ECM (Fig. 5A) The pancreas ECM was non-cytotoxic after 14

days and like other efficiently decellularized ECM scaffolds [48, 49], was not associated

with presence of multinucleate foreign body giant cells or other pathological signs of the

foreign body response (Fig. 5B). Furthermore, active angiogenesis was a feature of the

decellularized pancreas implantation site (Fig. 5B, arrows). These preliminary results

suggest that the perfusion-decellularized pancreas ECM described herein represents a

cytocompatible scaffold capable of integrating within host tissue.

3.5 Recellularization of decellularized pancreatic scaffold

To evaluate the potential of decellularized pancreas as a scaffold for pancreatic tissue

engineering, it was recellularized using relevant pancreatic cell lines. We first explored

the effect of two dimensional (2D) slices of decellularized pancreatic matrix as a

substrate for cell culture (Fig. 6A). Decellularized pancreatic matrix was cut into slices of

about 10-20 µm thickness, as evaluated by multi-photon microscopy (Fig. 6B). MIN-6

cells (25,000 cells/cm2) were seeded on the 2D slice and cultured for 5 days. After 5

days, MIN-6 cells were found attached and spread on the surface of 2D slice (Fig. 6C).

To evaluate whole organ recellularization potential, endocrine β cell line, MIN-6 cells

(30x106) were first seeded into the decellularized pancreas via hepatic portal vein by

multistep infusion technique following the seeding strategy described for liver

recellularization [20, 21]. Approximately 10x106 cells were introduced at each step, for a

total of 3 steps, with 20-min intervals between each step. Upon cell seeding, a uniform

color change was observed throughout the whole decellularized pancreas, indicating a

homogenous cell delivery (Fig. 7A-B). The repopulated pancreas was then maintained

in static culture for 5 days with culture media changed by perfusion through the

pancreas every day. After 5 days, the reconstructed pancreas was analyzed for

engraftment, survival and functionality by IHC. From the histology, MIN-6 cells appeared

to be engrafted both surrounding the parenchymal region and around the larger vessels

(Fig. 7C). TUNEL staining detected only minimal apoptosis (<18%), suggesting that the

3D decellularized pancreas scaffold is cytocompatible and supportive of cell growth

(Fig. 7D). Furthermore, these engrafted cells maintained their insulin expression over 5

days as evidenced by positive C-peptide staining (Fig. 7F).

The native pancreas is a dual-functioning glandular organ comprised and capable of

both endocrine and exocrine tissue and function, to this end, next the decellularized

pancreas was reconstructed with both the endocrine β cell line (MIN-6) and exocrine

acinar cell line (AR42J) by simultaneous seeding. The β cells were perfused through the

hepatic portal vein as before, while the acinar cells were perfused through the

pancreatic duct (Fig 8A). The two cell types were seeded in sequence, with the hepatic

duct perfusion of the MIN6 cells preceding the pancreatic duct perfusion of acinar

AR42J cells. This seeding strategy attempts to retain the cellular composition of native

pancreas and the anatomical proximity of the cells to different secretory locations: (i.e.;

endocrine β cells – vasculature; exocrine acinar cells – ductal system). As before, the

dual cell-seeded pancreas constructs was maintained for up to 5 days in static culture.

At day 5, IHC analysis showed that both cell types attached and localized to their

specific topographical locations as a result of their different cell seeding route (Fig. 8B).

Immunofluorescence staining confirmed the expression of C-peptide, as an analogue of

insulin marker identifying endocrine β-cells and amylase as a digestive enzyme

identifying exocrine acinar-cells (Fig. 8B, C). Most C-peptide+ cells were localized to the

luminal spaces of large vessels and parenchymal regions similar to that observed in

single cell reconstructed pancreas. Distinct from the localization of C-peptide+ cells,

tubular ductal spaces lined with acinar cells expressing amylase were observed (Fig.

8C). No inter-dispersed population of the two cell types were observed within the

engraftment region, suggesting the preservation of the distinct architecture of

vasculature and ductal structure (Fig. 8C). These results indicate that the decellularized

pancreas is capable of supporting multiple pancreatic cell types and this co-seeding

strategy allows the reconstitution of major cellular constituents as in the native

pancreas.

3.6 Modulation of β-cell insulin gene expression by native pancreatic ECM

Both primary pancreatic islets and individual isolated pancreatic β-cells have been

demonstrated to have better in vitro function when cultured on ECM-derived substrates

[37, 39-41, 50] and purified ECM proteins [15, 38, 51, 52]. Having confirmed

engraftment and survival of the relevant pancreatic cell types in the native pancreatic

scaffold, the contribution of this pancreas-derived ECM scaffold on cell function was

next evaluated by the insulin gene expression level. For comparative evaluation, control

cultures of MIN-6 cells (25,000 cells/cm2) were plated on surfaces with the following

ECM proteins: collagen I, collagen type IV, fibronectin, and growth factor-reduced

(GFR) Matrigel. In addition, the MIN-6 cells were also cultured on 2D slice

configurations of pancreatic ECM for comparison between the effects of 2D versus 3D

culture configurations. Upon plating, the MIN-6 cells readily attached to all tested

substrates and remained viable over the culture period (data not shown). The cells were

cultured for 5 days, after which the cell lysate was analyzed for insulin gene expression

(ins1 and ins2 expression) by qRT-PCR (Fig. 9). Quantitative RT-PCR of MIN-6

demonstrated significantly higher ins1 and ins2 gene expression when cultured on

these ECM proteins and pancreatic ECM scaffold (P<0.05) compared to control

(untreated plate) with the exception of collagen I gel. Among the ECM proteins tested,

collagen IV and fibronectin substrates induced similar insulin gene expression levels.

Matrigel promoted the highest insulin gene expression (6.9-fold ins1 and 4.1-fold ins2).

MIN-6 seeded on 3D pancreatic ECM exhibited higher insulin gene expression than 2D

pancreatic ECM. Furthermore, 3D pancreatic ECM also demonstrated higher insulin

gene expression when compared to single ECM proteins such as collagen IV and

fibronectin but displayed similar level of insulin gene expression with the Matrigel coated

surface (no significant difference). These results clearly demonstrate that β-cells interact

closely with the ECM substrates, and that interaction with specific substrates can

significantly affect insulin gene expression. The native organ derived of decellularized

pancreas was found to have a strong positive attribute to ins1 and ins2 gene

expression, comparable to Matrigel coating and significantly higher than other tested

single purified molecules.

4. DISCUSSION

The production of an intact acellular organ such as the pancreas by perfusion-

decellularization offers a promising alternate approach for pancreatic tissue engineering

and functional organ replacement. Herein, we demonstrate that perfusion-

decellularization of whole pancreas results in the generation of a natural pancreas ECM

scaffold with a perfusable vascular tree, ductal network and intact 3D architecture,

which acts as a suitable template for pancreatic tissue engineering and whole organ

regeneration. Comprehensive characterization of the decellularized pancreatic matrix

showed the preservation of complex multi-faceted ECM proteins, 3D spatial orientation

and microstructure, and relative stiffness of native pancreas. Decellularized pancreatic

matrix was non-cytotoxic, and promoted angiogenesis when subcutaneously implanted

in a mouse model. Repopulation of decellularized pancreas with pancreatic cells

showed the cytocompatibility of the scaffold. The recellularized constructs maintained

their respective phenotypic expression (β-cells: C-peptide; acinar cells: Amylase) after 5

days of culture. Finally, we demonstrated that the decellularized pancreas matrix is

supportive of β-cell function, as evidenced by the strong up-regulation of insulin gene

expression.

The objectives for successful decellularization are 1) complete or near complete

removal of cellular material, and 2) preservation of ECM composition [53]. Commonly

reported decellularization protocols require mechanical agitation or freezing and thawing

which may take up to days or weeks to remove all cellular materials [54-56]. Such

protocols do not guarantee preservation of the ECM microstructure, which has been

shown to be instrumental for the generation of functional tissue engineered constructs

[57]. In contrast, our approach of whole organ perfusion decellularization reduces the

diffusion distance required for decellularization agents to reach the cells and facilitates

removal of the cellular material from the tissue by convective transport [53, 58, 59]. This

technique allowed the efficient generation of acellular scaffold with preserved ECM, 3D

architecture and perfusable network resembling a native organ (approximately 325 min

as shown in our data Fig. 1D). The resulting decellularized pancreas met the stringent

requirement to define a successful decellularization [48] - absence of nuclear material

with DAPI and H&E staining, and retained only less than 50ng dsDNA per mg ECM dry

weight as shown by quantitative DNA measurement (Fig. 1E-G). This requirement is

crucial because residual DNA fragments in decellularized ECM have shown to lead to

cytocompatibility issues in vitro and adverse immunological response upon implantation

[49, 60, 61]. We and other groups have tried decellularization using similar strategies on

other circulatory networks to reduce diffusion distance (e.g., bile duct in liver, pancreatic

duct in pancreas) but it was much less effective likely due to incomplete distribution of a

conduit network to reach all the cells throughout the target organ.

Another important consideration for organ decellularization is minimizing the

undesirable alteration and loss of biologically active ECM components. Decellularized

pancreas scaffolds demonstrated maintenance of key ECM proteins in adult pancreas

[46, 47], including collagen I, collagen IV, laminin, and fibronectin, as well as sulfated

GAG. Most of the ECM proteins retained their physiological organization after

decellularization as noted by IHC (Fig. 2A). This finding was further substantiated by

mass spectrometry analysis to comprehensively characterize the proteome of the

decellularized pancreatic ECM. Retention of ECM protein/glycan compositions is

typically characterized by IHC; this approach is however limited by its robustness and

only confined to a small number of ECM candidates studied. Proteomic analysis by

mass spectrometry is unbiased and does not require specific probes. Though our

analysis was qualitative, notably all the top scoring proteins belonged to the ECM family

(supplemental table 1). These identifications have a high number of peptide spectral

counts providing rough measures of protein abundance.

Biomechanical evaluation of engineered scaffolds and decellularized constructs is an

important assessment for the rate of preservation of functional integrity. Numerous

studies have shown that biomechanical properties change after decellularization of

tissues [28, 62, 63]. By using AFM, we measured the stiffness of the decellularized

pancreas ECM. Our results demonstrated that Young’s modulus of decellularized

pancreas is on average 3 times greater than the native pancreas (P<0.01, Fig. 4A, B).

This is partially attributed to the loss of GAG content from the decellularization protocol

(Fig. 2D). GAG proteins have a myriad of biological functions and some of them are

associated with structural and biomechanical properties. GAGs possess a fixed

negative charge that renders them hydrophilic and attracts water into the tissue causing

an osmotic swelling, which in turn could contribute to tissue physical properties.

Removal of GAG side chains by heparitinase/chondroitinase treatment has been shown

to increase stiffness of basement membrane of tissue [11]. Although our result showed

a higher Young’s modulus in decellularized pancreas than native pancreas, it is still well

within the mechanical property range of “soft tissue” [64, 65]. While the magnitude of

increase depends on the specific organ under study, some studies have shown that a

slight alteration of tissue biomechanical properties was not associated with any

detrimental effect on cellular functionality. For instance, successful repopulation of a

cardiac matrix was demonstrated despite a higher tangential modulus was detected

within the decellularized heart when compared to native rat ventricles [16], which is

consistent with our findings.

Recent studies have suggested that the ECM composition and expression pattern are

specific to a given anatomical location to direct or support site-appropriate cell

attachment and function [25, 66]. In the native pancreas, the primary functional units of

the pancreas divide easily into endocrine cells and exocrine cells. The endocrine cells

are arranged mainly in groups as islets of Langerhans and secrete different

polypeptides delivered to other parts of the body via the vasculature. In contrast, the

secretions of acinar exocrine cells are carried away through the ductal system. To

reconstitute the major functional processes in pancreas, we utilized these unique

differential secretory channels to deliver two major pancreatic cell types – endocrine (β-

cells) and exocrine (acinar cells) – in close proximity to their respective native niches.

Our result showed that both cells types retained their functionality and distinct location

upon recellularization (Fig. 8B, C).

Reestablishment of a critical ECM-β-cells interaction has been shown to improve β-cells

survival and functions [15, 38, 40, 50-52]. In this study, we evaluated the effect of

substrate on β-cell function. Insulin gene expression of MIN-6 cultured on Matrigel was

found to be significantly higher than coated single purified proteins. This finding

indicates that β-cells are responsive to ECM substrate differences. Hence it was

expected that native pancreatic ECM scaffold with preserved native ECM milieu will also

likely enhance insulin gene expression. Consistent to our hypothesis, ins1 and ins2

expression of MIN-6 cultured on decellularized pancreas 3D scaffold was higher than

single purified molecules (P<0.05, Fig. 9) and comparable to Matrigel (no statistical

difference). However, 2D substrates of pancreatic ECM only showed similar level of

insulin gene expression with single purified molecules. This is likely due to the reason

that pancreatic cell niche in vivo is a 3D environment and previous studies have shown

that cell-matrix interaction in 3D matrices are more favorable from those on 2D

substrates [67, 68]. Our result was supportive of this previous finding with the 3D

pancreatic scaffold demonstrating better support of β-cell function. The high insulin

gene expression observed when β-cells are cultured on Matrigel suggests the benefits

of synergistic effects from multi-faceted ECM proteins. Similarly, decellularized

pancreas scaffold also possesses multiplex ECM composition and the effects are

proving to be similar when compared to Matrigel. Even though Matrigel is widely used in

various tissue engineering studies, it is limited in its translational potential due to its

tumor cell origin. In contrast, decellularized xenogenic sources of ECM scaffolds are

typically regulated by the FDA as medical device and currently in wide clinical use [69].

Hence, the development of a bioscaffold using decellularized pancreatic ECM will

provide a better clinical translation opportunity for pancreatic tissue engineering and

regenerative medicine applications.

5. CONCLUSIONS

This report presents the first step toward successful decellularization and 3D

reconstruction of whole pancreas. Thorough characterization revealed that the

decellularized pancreas retained its native 3D architecture, vasculature and ductal

channels along with important ECM compositions. The preserved pancreatic ECM

scaffold was cytocompatible, supportive of representative pancreatic cell types, and

enhanced insulin function when seeded with β-cells. Such native organ derived scaffold

is likely to have a great impact in pancreatic tissue engineering, by providing a niche

microenvironment for pancreatic cell types and even stem/progenitor cells.

ACKNOWLEDGEMENTS

We acknowledge our funding source for the generous support: NIH New Innovator

Award DP2 116520. We thank Jonathon Franks from Center of Biological Imaging

University of Pittsburgh for the technical assistance with SEM and TEM, and Timothy

Maul for his assistance with bovine pancreas procurement.

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FIGURE LEGENDS

Figure 1. Perfusion-decellularization of murine pancreas. (A-D) Panel images depict gradual change of color from

perfusion-decellularization of mouse pancreas with 0.5% SDS. Resulting decellularized pancreas after 325 min (D)

appeared translucent. (E) Histological comparison of native and decellularized pancreas by H&E staining showed

removal of cells. (F) IHC evaluation of native and decellularized pancreas depict presence of major pancreatic

constituents (C-peptide – red, and Amylase – green) in native pancreas (left) but absence in decellularized pancreas

(right). (G) DNA quantification via picogreen analysis demonstrated less than 50ng/mg dry weight of DNA material

present in the decellularized pancreas.

Figure 2. ECM characterization of decellularized pancreas. (A) IHC staining showed the presence of major ECM

composition – Col I, Col IV, Fibronectin, laminin and phalloidin actin staining (green) in native pancreas. (B)

Comparison with decellularized pancreas, IHC staining demonstrated the corresponding ECM markers found in

native pancreas also preserved in decellularized markers but DAPI and actin cytoskeletal element were absent. This

indicates complete removal of cellular materials but preservation of important ECM proteins. (C) Alcian blue staining

qualitatively determined the retention of sGAG protein after decellularization. (D) Blyscan assays quantitatively

determined the sGAG retention in decellularized pancreas was on average 52% of that in native pancreas (P<0.05).

Figure 3. Ultrastructural characterization of decellularized pancreas. (A) Trypan blue dye infusion visualized the

progressive flow from large vessels to the fine blood vessel branches along the channels without leakage. Inset

shows light microscopy image of vasculature branches remained intact after decellularization. (B) TEM of

decellularized pancreas depicted the intact basement membrane (arrowhead points to the underlying basement

membrane). (C) TEM image of decellularized pancreas showed the interstitial space with organized fibers of collagen

(arrowhead). (D-E) SEM comparison of native and decellularized pancreas demonstrated preservation of 3D micro-

structure of pancreatic duct after decellularization. Inset shows the absence of lining ciliated microvilli cells from the

luminal surface of the duct after decellularization process. (F) SEM image of decellularized pancreas showed ECM

fibers within the parenchymal space. (G) Higher magnification of 3D meshwork showed a variety of fibers - large

bundle of Type I collagen (black arrowhead) associated with a variety of smaller fibers (white arrowhead). (H) SEM

image of decellularized pancreas demonstrated large/small fibers interlinked in a plane that forms a boundary such

as to previously occupied pancreatic parenchymal cells. (I) SEM image of decellularized pancreas demonstrated

scalloping appearance of the internal elastic lamina of an artery, indicating intact blood vessel (BV).

Figure 4. Biomechanical properties of decellularization pancreas. (A) Representative deflection plot from AFM

evaluation of native and decellularized pancreas shows a steeper slope than force curve of decellularized pancreas

from native pancreas, indicating higher Young’s modulus. (B) Bar graph representatives of AFM force curve shows

on average 3 times higher stiffness in the decellularized pancreas than native pancreas.

Figure 5. Biocompatibility of perfusion-decellularized pancreas ECM. A perfusion-decellularized pancreas ECM

construct was surgically implanted within a subcutaneous pocket in a C57Bl/6 mouse model. After 14 days Mason’s

trichrome staining of the implant site (A) showed the ECM scaffold (#) infiltrated with host mononuclear cells. H&E

staining (B) showed no signs of multinucleate giant cells and the presence of blood vessel (arrows). (Scale bars =

50µm, inset = low power magnification, inset scale bar = 200µm)

Figure 6. MIN-6 culture on 2D slice pancreatic matrix. (A) Diagram illustrates the generation of 2D pancreatic

slice. White dashed circle depict the cloning cylinder applied to pancreatic slice to consolidate seeded cells to

pancreatic substrate. (B) Multi-photon imaging on second harmonic generation (SHG) signal to visualize the collagen

fibers demonstrated that thickness of the 2D slice pancreatic matrix is about 10-20 µm. (C) H&E staining

demonstrated the attachment of MIN-6 cells on 2D pancreatic slice after 5 days of culture, and insulin expression (C-

peptide: green) was maintained.

Figure 7. Recellularization of decellularized pancreas. (A) Decellularized whole pancreas before cell seeding. (B)

Recellularization of the same pancreas via multi-step infusion of about 30 million cells via portal vein infusion. (C)

H&E staining of the recellularized pancreas after 5 days showed the engraftment of the seeded cells at surrounding

parenchymal region. (D) TUNEL staining of the recellularized construct showed less than 18% of apoptotic cells

(arrowhead), demonstrating the cytocompatibility of the scaffold. (E) Positive control of TUNEL staining by adding

DNAse to the consecutive sections of recellularized pancreas’s histological slide. (F) IHC of recellularized pancreas

with MIN-6 only showed C-peptide+ cells engraftment around the larger vessel (arrowhead).

Figure 8. Dual recellularization strategy with β- and acinar cell types. (A) Two different cell types (MIN-6 and

AR42J) were inoculated into the decellularized pancreas 3D scaffold utilizing two different seeding routes. (B) IHC of

recellularization with both MIN-6 and AR42J cell lines showed engraftment of both cell types. Both cell types

maintained their respective functional markers – MIN-6 (Cpeptide – green) and AR42J (Amylase – red). (C)

Vasculature and pancreatic duct situated next to each other with their respective seeded different population of cells

(vasculature-MIN6 and duct-AR42J). Inset shows single channel images. This indicates that preservation of

architecture of vasculature and ductal structure, hence different cell population were not inter-dispersed after seeding.

Figure 9. Comparison of ECM mediated insulin gene expression of MIN-6 cells. qPCR data demonstrated

modulation of MIN-6 cell insulin gene expression by different types of ECM protein. 3D Pancreatic ECM

demonstrated higher ins1 and ins2 genes expression than Collagen I, Collagen IV, Fibronectin and 2D pancreatic

slice ECM but only comparable to Matrigel. Normalization was performed to MIN-6 cells seeded on control non-tissue

culture treated plastic. * indicates P<0.05

TABLES

Table 1. Mass Spectrometry Composition Analysis

Protein Family Gene Description

Collagens Col6a3 Collagen alpha-3(VI) chain

Col1a2 Collagen alpha-2(I) chain

Col4a2 Collagen alpha-2(IV) chain

Col4a1 Collagen alpha-1(IV) chain

Col1a1 Collagen alpha-1(I) chain

Col5a2 Collagen alpha-2(V)

Col6a2 Collagen alpha-2(VI)

Col6a1 Collagen alpha-1(VI)

Col5a1 Collagen alpha-1(V) chain

Col3a1 Collagen alpha-1(III)

Col14a1 Collagen alpha-1(XIV) chain

Col6a6 Collagen alpha-6(VI) chain

Col5a3 Procollagen, type V, alpha 3

Col7a1 Col7a1 Collagen alpha-1(VII) chain

Gm7455 Collagen alpha-5(VI) chain

Col4a3 Collagen alpha-3(IV) chain

Col18a1 Collagen alpha-1(XVIII) chain

Laminin Lamb2 Laminin subunit beta-2

Lamc1 Laminin subunit gamma-1

Lama2 Laminin subunit alpha-2

Lama5 Laminin subunit alpha-5

Lamb1 Laminin subunit beta-1

Lama1 Laminin subunit alpha-1

Lama4 Laminin subunit alpha-4

Proteoglycans Hspg2 Basement membrane-specific heparan sulfate proteoglycan core protein

Agrn Agrin

Nidogens Nid1 Nidogen-1

Nid2 Nidogen-2

Elastin associated Fbn1 Fibrillin-1

Matricellular Tnxb Tenascin-X

SUPPLEMENTARY DATA

Figure S1. Decellularization of bovine pancreas. Panel images show progressive decellularization of bovine

pancreas over the course of 65 hours.

Supplemental Table 1

Accession DescriptionP11087 Collagen alpha-1(I) chain OS=Musmusculus GN=Col1a1 PE=1 SV=4 - [CO1A1_MOUSE]Q01149 Collagen alpha-2(I) chain OS=Musmusculus GN=Col1a2 PE=2 SV=2 - [CO1A2_MOUSE]P08122 Collagen alpha-2(IV) chain OS=Musmusculus GN=Col4a2 PE=2 SV=4 - [CO4A2_MOUSE]P08121 Collagen alpha-1(III) chain OS=Musmusculus GN=Col3a1 PE=2 SV=4 - [CO3A1_MOUSE]Q3U962 Collagen alpha-2(V) chain OS=Musmusculus GN=Col5a2 PE=1 SV=1 - [CO5A2_MOUSE]Q9JLI2 Collagen type V alpha 3 chain OS=Musmusculus GN=Col5a3 PE=2 SV=1 - [Q9JLI2_MOUSE]P02463 Collagen alpha-1(IV) chain OS=Musmusculus GN=Col4a1 PE=2 SV=4 - [CO4A1_MOUSE]P02535-3 Isoform 3 of Keratin, type I cytoskeletal 10 OS=Musmusculus GN=Krt10 - [K1C10_MOUSE]J3QQ16 Protein Col6a3 OS=Musmusculus GN=Col6a3 PE=4 SV=1 - [J3QQ16_MOUSE]E9PZ16 Endorepellin OS=Musmusculus GN=Hspg2 PE=4 SV=1 - [E9PZ16_MOUSE]P11679 Keratin, type II cytoskeletal 8 OS=Musmusculus GN=Krt8 PE=1 SV=4 - [K2C8_MOUSE]O88207 Collagen alpha-1(V) chain OS=Musmusculus GN=Col5a1 PE=2 SV=2 - [CO5A1_MOUSE]Q04857 Collagen alpha-1(VI) chain OS=Musmusculus GN=Col6a1 PE=2 SV=1 - [CO6A1_MOUSE]Q61292 Laminin subunit beta-2 OS=Musmusculus GN=Lamb2 PE=2 SV=2 - [LAMB2_MOUSE]F8VQJ3 Laminin subunit gamma-1 OS=Musmusculus GN=Lamc1 PE=4 SV=1 - [F8VQJ3_MOUSE]Q02788 Collagen alpha-2(VI) chain OS=Musmusculus GN=Col6a2 PE=2 SV=3 - [CO6A2_MOUSE]Q61001 Laminin subunit alpha-5 OS=Musmusculus GN=Lama5 PE=1 SV=4 - [LAMA5_MOUSE]E9Q1Z0 Protein 4732456N10Rik OS=Musmusculus GN=4732456N10Rik PE=3 SV=1 - [E9Q1Z0_MOUSE]A2AQ53 Fibrillin 1 OS=Musmusculus GN=Fbn1 PE=4 SV=1 - [A2AQ53_MOUSE]F8VQ43 Laminin subunit alpha-2 OS=Musmusculus GN=Lama2 PE=4 SV=1 - [F8VQ43_MOUSE]E9Q557 Desmoplakin OS=Musmusculus GN=Dsp PE=3 SV=1 - [DESP_MOUSE]E9Q6A6 Collagen alpha-6(VI) chain OS=Musmusculus GN=Col6a6 PE=4 SV=2 - [E9Q6A6_MOUSE]P11276 Fibronectin OS=Musmusculus GN=Fn1 PE=1 SV=4 - [FINC_MOUSE]E9Q3W4 Plectin OS=Musmusculus GN=Plec PE=4 SV=1 - [E9Q3W4_MOUSE]E9QPE7 Myosin-11 OS=Musmusculus GN=Myh11 PE=4 SV=1 - [E9QPE7_MOUSE]E9Q2T3 Protein Tnxb OS=Musmusculus GN=Tnxb PE=4 SV=1 - [E9Q2T3_MOUSE]O08638 Myosin-11 OS=Musmusculus GN=Myh11 PE=1 SV=1 - [MYH11_MOUSE]Q792Z1 MCG140784 OS=Musmusculus GN=Try10 PE=2 SV=1 - [Q792Z1_MOUSE]Q02257 Junction plakoglobin OS=Musmusculus GN=Jup PE=1 SV=3 - [PLAK_MOUSE]Q63ZW6 Col4a5 protein OS=Musmusculus GN=Col4a5 PE=2 SV=1 - [Q63ZW6_MOUSE]Q80X19-2 Isoform 2 of Collagen alpha-1(XIV) chain OS=Musmusculus GN=Col14a1 - [COEA1_MOUSE]P02469 Laminin subunit beta-1 OS=Musmusculus GN=Lamb1 PE=1 SV=3 - [LAMB1_MOUSE]Q9CPN9 Protein 2210010C04Rik OS=Musmusculus GN=2210010C04Rik PE=2 SV=1 - [Q9CPN9_MOUSE]B1AVK5 Procollagen type IV alpha 6 OS=Musmusculus GN=Col4a6 PE=4 SV=1 - [B1AVK5_MOUSE]P07356 Annexin A2 OS=Musmusculus GN=Anxa2 PE=1 SV=2 - [ANXA2_MOUSE]Q6IFZ8 MCG1050941 OS=Musmusculus GN=Gm5414 PE=2 SV=1 - [Q6IFZ8_MOUSE]A6H584 Collagen alpha-5(VI) chain OS=Musmusculus GN=Col6a5 PE=1 SV=2 - [CO6A5_MOUSE]P10493 Nidogen-1 OS=Musmusculus GN=Nid1 PE=1 SV=2 - [NID1_MOUSE]Q63870 Collagen alpha-1(VII) chain OS=Musmusculus GN=Col7a1 PE=2 SV=3 - [CO7A1_MOUSE]Q78PY7 Staphylococcal nuclease domain-containing protein 1 OS=Musmusculus GN=Snd1 PE=1 SV=1 - [SND1_MOUSE]P16546-2 Isoform 2 of Spectrin alpha chain, non-erythrocytic 1 OS=Musmusculus GN=Sptan1 - [SPTN1_MOUSE]

E9Q1F2 Actin, cytoplasmic 1, N-terminally processed OS=Musmusculus GN=Actb PE=3 SV=1 - [E9Q1F2_MOUSE]Q8VDD5 Myosin-9 OS=Musmusculus GN=Myh9 PE=1 SV=4 - [MYH9_MOUSE]Q9QZR9 Collagen alpha-4(IV) chain OS=Musmusculus GN=Col4a4 PE=2 SV=1 - [CO4A4_MOUSE]P21981 Protein-glutamine gamma-glutamyltransferase 2 OS=Musmusculus GN=Tgm2 PE=1 SV=4 - [TGM2_MOUSE]Q61495 Desmoglein-1-alpha OS=Musmusculus GN=Dsg1a PE=2 SV=2 - [DSG1A_MOUSE]A1BN54 Alpha actinin 1a OS=Musmusculus GN=Actn1 PE=2 SV=1 - [A1BN54_MOUSE]P63268 Actin, gamma-enteric smooth muscle OS=Musmusculus GN=Actg2 PE=2 SV=1 - [ACTH_MOUSE]O88322 Nidogen-2 OS=Musmusculus GN=Nid2 PE=1 SV=2 - [NID2_MOUSE]P62806 Histone H4 OS=Musmusculus GN=Hist1h4a PE=1 SV=2 - [H4_MOUSE]P68373 Tubulin alpha-1C chain OS=Musmusculus GN=Tuba1c PE=1 SV=1 - [TBA1C_MOUSE]A2AJY2 Collagen alpha-1(XV) chain OS=Musmusculus GN=Col15a1 PE=4 SV=1 - [A2AJY2_MOUSE]P00688 Pancreatic alpha-amylase OS=Musmusculus GN=Amy2 PE=1 SV=2 - [AMYP_MOUSE]E9Q5F6 Ubiquitin-related 2 (Fragment) OS=Musmusculus GN=Ubc PE=4 SV=1 - [E9Q5F6_MOUSE]P07901 Heat shock protein HSP 90-alpha OS=Musmusculus GN=Hsp90aa1 PE=1 SV=4 - [HS90A_MOUSE]P97927 Laminin subunit alpha-4 OS=Musmusculus GN=Lama4 PE=1 SV=2 - [LAMA4_MOUSE]Q9Z1Z0-3 Isoform 3 of General vesicular transport factor p115 OS=Musmusculus GN=Uso1 - [USO1_MOUSE]Q61879 Myosin-10 OS=Musmusculus GN=Myh10 PE=1 SV=2 - [MYH10_MOUSE]Q9JIF7 Coatomer subunit beta OS=Musmusculus GN=Copb1 PE=1 SV=1 - [COPB_MOUSE]P14148 60S ribosomal protein L7 OS=Musmusculus GN=Rpl7 PE=2 SV=2 - [RL7_MOUSE]Q61753 D-3-phosphoglycerate dehydrogenase OS=Musmusculus GN=Phgdh PE=1 SV=3 - [SERA_MOUSE]Q8BU30 Isoleucine--tRNA ligase, cytoplasmic OS=Musmusculus GN=Iars PE=2 SV=2 - [SYIC_MOUSE]P17897 Lysozyme C-1 OS=Musmusculus GN=Lyz1 PE=1 SV=1 - [LYZ1_MOUSE]Q9WTI7-4 Isoform 4 of Unconventional myosin-Ic OS=Musmusculus GN=Myo1c - [MYO1C_MOUSE]P46978 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit STT3A OS=Musmusculus GN=Stt3a PE=1 SV=1 -

[STT3A_MOUSE]E9PVA8 Protein Gcn1l1 OS=Musmusculus GN=Gcn1l1 PE=4 SV=1 - [E9PVA8_MOUSE]B7FAV1 Filamin, alpha (Fragment) OS=Musmusculus GN=Flna PE=4 SV=1 - [B7FAV1_MOUSE]F8VQ40 Laminin subunit alpha-1 OS=Musmusculus GN=Lama1 PE=4 SV=1 - [F8VQ40_MOUSE]Q9EST1 Gasdermin-A OS=Musmusculus GN=Gsdma PE=2 SV=1 - [GSDMA_MOUSE]E9Q3D6 Heat shock protein HSP 90-beta (Fragment) OS=Musmusculus GN=Hsp90ab1 PE=4 SV=1 - [E9Q3D6_MOUSE]P10126 Elongation factor 1-alpha 1 OS=Musmusculus GN=Eef1a1 PE=1 SV=3 - [EF1A1_MOUSE]P17809 Solute carrier family 2, facilitated glucose transporter member 1 OS=Musmusculus GN=Slc2a1 PE=1 SV=4 - [GTR1_MOUSE]Q8VDN2 Sodium/potassium-transporting ATPase subunit alpha-1 OS=Musmusculus GN=Atp1a1 PE=1 SV=1 - [AT1A1_MOUSE]P99024 Tubulin beta-5 chain OS=Musmusculus GN=Tubb5 PE=1 SV=1 - [TBB5_MOUSE]B7ZN27 Cad protein OS=Musmusculus GN=Cad PE=2 SV=1 - [B7ZN27_MOUSE]P22892 AP-1 complex subunit gamma-1 OS=Musmusculus GN=Ap1g1 PE=1 SV=3 - [AP1G1_MOUSE]P10852 4F2 cell-surface antigen heavy chain OS=Musmusculus GN=Slc3a2 PE=1 SV=1 - [4F2_MOUSE]Q61555 Fibrillin-2 OS=Musmusculus GN=Fbn2 PE=1 SV=2 - [FBN2_MOUSE]P58252 Elongation factor 2 OS=Musmusculus GN=Eef2 PE=1 SV=2 - [EF2_MOUSE]Q9D6F9 Tubulin beta-4A chain OS=Musmusculus GN=Tubb4a PE=1 SV=3 - [TBB4A_MOUSE]Q76MZ3 Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform OS=Musmusculus GN=Ppp2r1a PE=1

SV=3 - [2AAA_MOUSE]Q9QZS0 Collagen alpha-3(IV) chain OS=Musmusculus GN=Col4a3 PE=1 SV=2 - [CO4A3_MOUSE]P70168 Importin subunit beta-1 OS=Musmusculus GN=Kpnb1 PE=1 SV=2 - [IMB1_MOUSE]Q922D8 C-1-tetrahydrofolate synthase, cytoplasmic OS=Musmusculus GN=Mthfd1 PE=1 SV=4 - [C1TC_MOUSE]Q68FD5 Clathrin heavy chain 1 OS=Musmusculus GN=Cltc PE=1 SV=3 - [CLH_MOUSE]

Q02053 Ubiquitin-like modifier-activating enzyme 1 OS=Musmusculus GN=Uba1 PE=1 SV=1 - [UBA1_MOUSE]Q8BMJ2 Leucine--tRNA ligase, cytoplasmic OS=Musmusculus GN=Lars PE=2 SV=2 - [SYLC_MOUSE]P32037 Solute carrier family 2, facilitated glucose transporter member 3 OS=Musmusculus GN=Slc2a3 PE=1 SV=1 - [GTR3_MOUSE]Q9D0R2 Threonine--tRNA ligase, cytoplasmic OS=Musmusculus GN=Tars PE=1 SV=2 - [SYTC_MOUSE]D3YXT0 NADH dehydrogenase [ubiquinone] iron-sulfur protein 2, mitochondrial OS=Musmusculus GN=Ndufs2 PE=3 SV=1 -

[D3YXT0_MOUSE]P28481-2 Isoform 3 of Collagen alpha-1(II) chain OS=Musmusculus GN=Col2a1 - [CO2A1_MOUSE]Q3UQ28 Peroxidasin homolog OS=Musmusculus GN=Pxdn PE=2 SV=2 - [PXDN_MOUSE]F8WIT2 Annexin OS=Musmusculus GN=Anxa6 PE=3 SV=1 - [F8WIT2_MOUSE]Q504P4 Heat shock cognate 71 kDa protein OS=Musmusculus GN=Hspa8 PE=2 SV=1 - [Q504P4_MOUSE]B9EJ54 MCG21756, isoform CRA_b OS=Musmusculus GN=Nup205 PE=2 SV=1 - [B9EJ54_MOUSE]O70133 ATP-dependent RNA helicase A OS=Musmusculus GN=Dhx9 PE=1 SV=2 - [DHX9_MOUSE]Q3V3R1 Monofunctional C1-tetrahydrofolate synthase, mitochondrial OS=Musmusculus GN=Mthfd1l PE=1 SV=2 - [C1TM_MOUSE]P52480-2 Isoform M1 of Pyruvate kinase isozymes M1/M2 OS=Musmusculus GN=Pkm - [KPYM_MOUSE]P52431 DNA polymerase delta catalytic subunit OS=Musmusculus GN=Pold1 PE=1 SV=2 - [DPOD1_MOUSE]Q3TXS7 26S proteasome non-ATPase regulatory subunit 1 OS=Musmusculus GN=Psmd1 PE=1 SV=1 - [PSMD1_MOUSE]D3YWT0 Signal peptidase complex catalytic subunit SEC11A OS=Musmusculus GN=Sec11a PE=4 SV=1 - [D3YWT0_MOUSE]Q8BP47 Asparagine--tRNA ligase, cytoplasmic OS=Musmusculus GN=Nars PE=1 SV=2 - [SYNC_MOUSE]P39061-2 Isoform 3 of Collagen alpha-1(XVIII) chain OS=Musmusculus GN=Col18a1 - [COIA1_MOUSE]Q9JHU4 Cytoplasmic dynein 1 heavy chain 1 OS=Musmusculus GN=Dync1h1 PE=1 SV=2 - [DYHC1_MOUSE]F8WJL5 Glyceraldehyde-3-phosphate dehydrogenase OS=Musmusculus GN=Gapdh PE=3 SV=1 - [F8WJL5_MOUSE]Q6URW6-2 Isoform 2 of Myosin-14 OS=Musmusculus GN=Myh14 - [MYH14_MOUSE]O54734 Dolichyl-diphosphooligosaccharide--protein glycosyltransferase 48 kDa subunit OS=Musmusculus GN=Ddost PE=1 SV=2 -

[OST48_MOUSE]P09405 Nucleolin OS=Musmusculus GN=Ncl PE=1 SV=2 - [NUCL_MOUSE]A2AVJ7 Ribosome binding protein 1 OS=Musmusculus GN=Rrbp1 PE=4 SV=1 - [A2AVJ7_MOUSE]Q9JLI8-2 Isoform 2 of Squamous cell carcinoma antigen recognized by T-cells 3 OS=Musmusculus GN=Sart3 - [SART3_MOUSE]Q7TMB8-2 Isoform 2 of Cytoplasmic FMR1-interacting protein 1 OS=Musmusculus GN=Cyfip1 - [CYFP1_MOUSE]Q9EPL8 Importin-7 OS=Musmusculus GN=Ipo7 PE=1 SV=2 - [IPO7_MOUSE]P10107 Annexin A1 OS=Musmusculus GN=Anxa1 PE=1 SV=2 - [ANXA1_MOUSE]E9QKZ2 Importin-9 OS=Musmusculus GN=Ipo9 PE=4 SV=1 - [E9QKZ2_MOUSE]